BRPI0901758A2 - use of telokinobufagin as an analgesic in the treatment of acute and chronic pain; pharmaceutical composition containing telokinobufagin and its use - Google Patents

Abstract

USE OF TELOCINOBUFAGINE AS ANALGESIC IN THE TREATMENT OF ACUTE AND CHRONIC PAIN; PHARMACEUTICAL COMPOSITION CONTAINING TELOCINOBUFAGIN AND USE OF THE SAME. The present invention is directed to the use of telokinobufagin, or pharmaceutically acceptable derivatives thereof, in the manufacture of a medicament for the treatment or prevention of pain in humans or animals. Additionally, the present invention is directed to the use of telokinobufagin as an analgesic for the treatment or prevention of acute and chronic pain. The present invention also relates to a pharmaceutical composition containing an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable excipients. The present invention also provides a method for inducing analgesia in response to acute and chronic pain which comprises administering an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, to humans or animals. Telokinobufagin, according to the results of in vivo tests, has a greater potency than that of morphine, evidenced when the comparison of the results is performed considering the molar masses of the two substances, without, however, presenting the known side effects of opioids, a Since the analgesic effects of TCB are independent of the opioid system. In addition, in vivo and in vitro tests have shown that TCB does not have cardiotoxicity.

Description

"USE OF TELOCINOBUFAGINE AS ANALGESIC IN THE TREATMENT OF ACUTE AND CHRONIC PAIN; CONTENDOTELOCINOBUFAGINE PHARMACEUTICAL COMPOSITION AND USE OF THE SAME".

The present invention relates to the field of analgesia and treatment or prevention of pain.

The present invention is directed to the use of detelokinobufagin (TCB), or pharmaceutically acceptable derivatives thereof, in the manufacture of a medicament for treating or preventing pain in humans or animals.

Additionally, the present invention is directed to the use of telokinobufin, or pharmaceutically acceptable derivatives thereof, as an analgesic in acute pain. The present invention also relates to a pharmaceutical composition containing an effective amount of detelokinobufagin, or pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable excipients.

According to another embodiment of the present invention there is provided a method for treating or preventing acute and chronic pain comprising administering an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, to humans or animals in need of said treatment.

According to the International Association for the Study of Pain (IASP), ador is characterized as "an unpleasant sensory and emotional experience brought on by actual or potential tissue injury or delineated in terms of such injury" [Loeserf JD, Melzack , R. 1979. Pain: an overview. Lancet 353: 1607-1609]. Physiologically it functions as an important warning signal triggering defense and preservation reactions.

Pain is a symptom of many clinical disorders and affects a large part of the population. It is generally responsible for a significant part of the demand for health services and is a multifactorial phenomenon involving psychosocial, behavioral and pathophysiological processes.

In addition to involving a potentially harmful stimulus, ador has an individual connotation and is represented by subjective experience, including affective and emotional behaviors, which amplify or diminish the painful sensation [Almeida, R. f. Roizenblattr S., Tufik, S. 2004. Afferentpain pathways: a neuroanatomical review. Brain Res 1000: 40-56].

To facilitate communication and interpretation of data, the IASP developed a taxonomy that classifies pain into five major items according to the region affected, the system involved, the temporal characteristic of pain, the pain intensity reported by the patient, and the etiology of pain.

The affected region refers to the head, face, mouth, cervical region, shoulders and upper limbs, thoracic region, abdominal region, lumbosacral spine, coccyx, lower limbs, pelvic region, perineal region, egenital anal.

The system involved refers to the nervous system (central, peripheral and / or neurovegetative), psychological and social factors, respiratory and / or cardiovascular, musculoskeletal and / or glandular, gastrointestinal, genitourinary, other organs or viscera.

As for the temporal characteristics of pain, they may be continuous or almost continuous without fluctuations, continuous with exacerbations, recurrent with regularity, recurrent without irregularity, paroxysmal and combinations.

As for the intensity, the pain reported by the patient may be mild, moderate and intense.

Regarding the etiology, pain may be caused by congenital or genetic disorders, trauma, surgery or burns, infectious and / or parasitic disease, metabolic disease, inflammation, autoimmune disease, cancer, poisoning, metabolic disorders and disorders, degenerative symptoms, dysfunctional conditions, psychological changes and unknown cause.

In addition to these, pain is also classified as to duration or evolution, which can be: acute or chronic. The acute pain is of physiological character, is triggered by injury and has alert and defensive function. It is mediated by thermal, mechanical or chemical stimulation of donor receptors. It is usually brief in duration and may be of two types: neurogenic when provoked by peripheral nociceptive stimulation or inflammatory when provoked by inflammatory reaction. Therefore, acute pain has a well-determined cause-and-effect relationship, being characterized by serpontual, delimited and disappearing with resolution of the pathological process. Chronic pain is gradually disabling as it may persist over a long period of time, and in some cases is related to changes in central nociception mechanisms. It may exist or persist in the absence of actual injury, produce persistent changes in psychomotor (emotional) behavior and may lead to permanent physical and mental disability [Almeida, R. F. Roizenblatt, S., Tufik, S. 2004. Afferent pain pathways: aneuroanatomical review. Brain Res 1000: 40-56].

The most common chronic pains include cancer-associated pain, myofascial pain syndrome, headache, fibromyalgia, pain associated with rheumatoid arthritis, phantom pain, central and dorneuropathic pain syndromes [Ashburn, Μ. THE.; Staats, P. S. 1999. Managementof chronic pain. Lancet, v. 353, p. 1865-1869,].

Neuropathic pain is always chronic and results from neural injury or dysfunction of neural pain circuits. In these cases there is no tissue injury, but anomalous activation of neural pathways that lead to pain information. Patients describe such pain as burning, pin or needle piercing, electric shock or numbness. Dermatome-corresponding skin territories tend to occur and may not respond well to opioids. Psychogenic pains are generally chronic, associated with depressive and / or convergent symptoms and do not fit the mechanisms described above.

In 1994, the IASP proposed the definition of neuropathic pain as pain of neural origin due to a primary injury or nervous system dysfunction [Merskey H, BogdukN. 1994. Classification of Chronic Painr 2nd edn. Seattle: IASP Press, 394]. Neuropathic pain may be caused by lesions of the roots and peripheral nerves, spinal cord, brainstem and brain. It is a complex and heterogeneous entity, with signs and symptoms that may fluctuate in intensity over time. Neuropathic pain is associated with various pathological conditions (Table 1) and is clinically associated with several other symptoms.

Table 1: Traditional etiological classification of dorneuropathic.

<table> table see original document page 5 </column> </row> <table>

Neuropathic pain is often described as a burning or stinging tendocharacter and is often associated with allodynia and / or hyperalgesia. Allodynia is defined as pain in response to a stimulus that normally does not cause pain (non-injurious) and hyperalgesia with exacerbated sensitivity to a stimulus that is painful (injurious).

Pain is a very prevalent symptom in patients with neoplasia. It affects about 50% to 70% of patients with general neoplasia and up to 90% of those with more advanced stages of the disease. The pain associated with neoplasms is usually intense and results from mechanisms such as: invasion and / or compression of nerves, bones and soft tissues, obstruction and / or distension of hollow viscera; Paraneoplastic neuropathies and toxic or inflammatory neuropathies resulting from surgery, chemotherapy or radiotherapy. More than two thirds of cancer-related pain results from direct tumor aggression and a fourth result from antineoplastic treatment. Such pains are multifaceted and can be classified as acute, chronic, nociceptive, neuropathic or psychogenic.

Nociceptive pain (from Latin nocere, which means bad) is acute or chronic and results from tissue damage by trauma, inflammation or compression (herniations and tumors). These processes promote physiological stimulation of free nerve determinations in any innervated territories. Its intensity is proportional to the degree of receptor activation and is generally well controlled with opioids. [ESMO. Management of cancer pain: ESMO Clinical Recommendations. Annals of Oncology. 2007. 18 (Suppl 2): ii92-ii94. Polomano, R. C. Farrar, J. T, 2006. Pain and neuropathy in cancer survivors. Am. J. Nurs., 106 (3 Suppl): 39-47].

There are numerous effective medications for acute donor treatment caused by various etiologies such as non-steroidal antiinflammatory opioids (NSAIDs). For dorneuropathic treatment options are less effective or less tolerable. Due to their distinct pathophysiological mechanisms, agents useful in treating other types of pain have only reduced efficacy in treating neuropathic donors.

Morphine, for example, is widely used for administration in severe acute pain due to its global availability, extensive clinical experience, significant pharmacokinetic and pharmacodynamic data, low cost and various sustained release formulations. However, the incidence and severity of adverse effects, among others, the phenomenon of tolerance (need for increasing doses to maintain the effect), dependence (physical and emotional need for use), nausea, constipation, pruritus, drowsiness and respiratory depression may be limiting factors. of its therapeutic use.

In particular, the efficacy of opioids in the treatment of neuropathic pain is decreased compared to the treatment of inflammatory pain [Bridgesf D., Thompson, S. W. Ν. Ricef A. S.C. 2001. Mechanisms of neuropathic pain. British Journal of Anaesthesia 87 (1): 12-26] [Sindrup S. Η. , Jensenf T. S.1999. Efficacy of pharmacological treatments of neuropathicpain: an update effect related to mechanism of drug action.Pain; 83: 389-400].

The class of NSAIDs (non-steroidal anti-inflammatory drugs) is highly effective for the treatment of mild and moderate acute screen pain. The main mechanism by which NSAIDs exert an analgesic effect is by inhibiting the synthesis of certain prostaglandins or prostanoids. Prostanoid synthesis uses two distinct enzymes cyclooxygenases (COX): COX-I and COX-2. Traditional NSAIDs inhibit both enzymes. They may also inhibit other lipogenic enzymes, such as 5-lipooxygenase. Although NSAIDs are not addictive, they have significant toxic effects such as gastrointestinal injury, nephrotoxicity and inhibition of platelet aggregation.

Alternatively, the major classes of analgesic adjuvants currently used to assist in the management of neuropathic pain include antidepressants, anticonvulsants, local anesthetics, muscle relaxants, and sympatholytics, among others [Max, Μ. B. 1994. In: Progress in Pain Research and Management (Ed, Fields H.L., Liebskind J.C.) IASP Press, Seatfle, 229].

Some anticonvulsants used in neuropathic donor treatment are: a) carbamazepine, which has central and peripheral effects, but has a high incidence of adverse effects related to the central nervous system (blurred vision, dizziness, headache, mental confusion, nausea, etc.) and high potential. of interaction with other drugs; b) oxcarbazepine, which is safer than carbamazepine, however, can cause serious damage in patients with renal insufficiency, in addition to the reduction of noplasma sodium levels, leukopenia, thrombocytopenia and skin rash; c) phenytoin, which is more potent than carbamazepine, however, due to the phenomenon called orderzero kinetics, as serum concentration increases, the enzyme system responsible for drug metabolism becomes unsaturated, causing small increases in dose to produce large elevations in serum levels, with their undesirable side effects; d) Gabapentin, which is considered one of the safest pharmacokinetic drugs, however, because high doses are required for treatment, the cost is usually not feasible. Also, association with other drugs is usually necessary.

However, currently available treatments have limited efficacy (complete relief of the drug is rarely achieved) and there is still a high incidence of debilitating adverse effects such as those mentioned above.

Due to the complexity of the pathophysiology of pain, severe and chronic acute pain can hardly be effectively treated using a single agent. It is common in practice to associate different pharmacological agents with different mechanisms of action. One strategy, for example, involves the use of low opioid analgesic doses in combination with analgesic doses of one or more non-opioid drugs. A better analgesia profile is obtained with a lower incidence of adverse effects and intolerance, but involves the risks associated with multitherapy.

Additionally, several other drugs have been used in pain management, including neuroactive steroids, beta-adrenergic agonists, selective prostanoid pain receptor antagonists; NMDA antagonists; neuronal nicotinic doreceptor agonists; decalcium channel antagonists; 5-HTserotonin receptor agonists (1B / 1D), cannabinoid agonists; superoxide dismutase mimetics; p3 8 MAP kinase inhibitor; triptans, TRPV1 agonists, ketamine, NK1 receptor antagonists; gabapentinoids, glycine receptor antagonists.

Thus, it is possible to state that the treatment of dorneuropathic pain is usually difficult and usually disappointing. For these reasons, in addition to medications, patients with neuropathic pain often require, for example, neurosurgical measures, anesthetic blocks, regional sympatholytic infusion, infiltrations, electrode neurostimulation, acupuncture, intractable drug infusions, physiotherapy, psychological and occupational therapies that help the patient. to live with the pain.

Thus, there is still a real need for effective and safe treatments to solve the problem of pain, especially acute and chronic pain. It is in this context that the present invention describes the use of datelokinobufagin as an effective and safe analgesic for treating or preventing pain, whether acute or chronic.

Telokinobufagin (TCB), whose chemical name is (3β, 5β) -3, 5, 14-trihydroxy-Bufa-2 0,22-dienolide, belongs to the classics steroid bufadienolides and its registration number CAS 472-26-4. Telokinobufagin has the molecular formula C24H34O5, molecular weight 402.52 and its structural formula (Formula I) is as follows:

<formula> formula see original document page 10 </formula>

Formula I - Telokinobufagin

Telokinobufagin was first described by Meyerem 194 9 together with other bufadienolides (cinobufagin, bufalin, bufotaline and cinobufotalin), all isolated from frog venom and having digitalis properties. The compounds have been isolated from the traditional prepared Ch'an Sul tea containing frog venom (Ch'an Sur in Chinese and Senso in Japanese) [Meyer, K. 1949. Cardioactivetoad poisons (bufogenin). I. Isolation of the cardioactive principles of Ch'an Su (sense). Pharmaceutica ActaHelvetiaer 24: 222-46 and Cardiac-active toad poisons (bufogenins). II. Constitution of Bufalin. Meyer, K. 1949. Helvetiea Chimica Aetaf 32: 1238-45].

Its synthesis was first presented by Pettitem 1974, who described a synthesis route starting from dadigitoxygenin to bufalin and scilarenin until it reached telokinobufagin. As scilarenin is available from naturalproscilaridine A glycoside, the described synthesis allows easier access to telokinobufagin and related substances [Pettit, G.R., Kamanor Y. Bufadienolides. 27. 1974. Synthesis of Telocinobufagin. J. Org.Chem., 39 (17): 2632-2634].

The term bufadienolide refers to the class of steroids that occur in venom and amphibian skin, and in some plant tissue. Vegetable, fruits and snakes have also been isolated. Bufadienolides consist of a steroid ring attached to the six-membered lactone group.

In the animal kingdom, bufadienolids are widely diffused in the Bufonidae family and several of them have already been isolated from the genus Bufo, mostly derived from steroids. For example, Rossi et alidentified in the venom of the Bufoparacnemis parotid glands the following compounds: sistoterol, argentinogenin, bufalin, bufotalinin, gambufotalin, helebrigenin, marleobufogenin, resinobufogenin and telokinobufagin. [Rossi H., Blumenthal, Ε. Ε A., Jared, C. 1997. Bufodienolides from the venom of Bufo paracnemis [anphibia, anura, bufonidae]. AnaisAssoc.Bras. Chem. 46 (1): 21-26].

The medical-pharmaceutical potential of products derived from toxic secretions of the genus Bufo is well known. Historical reports reveal the therapeutic use of a commercial preparation obtained from the dry poison of frogBufo gargarizans, the secular Ch'an Su (or Senso). This is probably the first and oldest use of an animal source poison in therapy. Their widely diversified jobs involve healing skin wounds, controlling mucosal bleeding, and colds. It has also been used in the treatment of heart disease and is known to have no advantages over digitalis [Hong, Zr Chanf Kr Yeung, H.W. 1992. Simultaneous determination of Bufadienolides. In: traditional Chinese medicine preparation, Liu-Shen-wan, byliquid chromatography. J. Pharm. Pharmacol, 44: 1023-1026].

Bufogenins and bufotoxins act similarly to digitalis extracts and are cardiotoxic to the mammalian concentration found in the venom. They are inhibitors of Na + K + ATPase enzyme and are involved in water and salt homeostasis in frog skin. These steroids increase blood pressure and stimulate the smooth muscle of some mammals even at diluted concentrations [Toledo, R. C; Jaredr C. 1995. Granular cutaneous glands and amphibianvenoms. Comp. Biochem. Physiol. 111 (1): 1-29].

International patent application W00214343 A1 (Novolanet alr 2 002) describes the use of bufadienolides in a pharmaceutical preparation with immunomodulatory activity, especially for immunosuppression, antiinflammatory and / or, in the case of non-cardioactive, antiproliferative bufadienolides. The paper suggests a clinical protocol for the treatment of rheumatic pain in humans, in which patients with inflammatory forms of rheumatism would receive injections of a bufadienolide, preferably Proscillaridin A (0.1 - 3 mg), into inflamed joints or tissues, but no biological or clinical results are shown. clinical support for reducing inflammation and pain.

The object of the present invention is to provide alternatives for the treatment or prevention of pain. Accordingly, the present invention proposes the use of telokinobufagin as an effective and safe analgesic for the treatment or prevention of acute and chronic pain.

According to an embodiment of the present invention, the use of telokinobufagine (Formula I) and pharmaceutically acceptable derivatives thereof is described in the manufacture of a medicament for treating or preventing pain. More specifically, the present invention describes the use of detelokinobufagin and pharmaceutically acceptable damesma derivatives for the treatment or prevention of acute screen pain in humans or animals.

According to an embodiment of the present invention there is described the use of a pharmaceutical composition comprising: (a) telokinobufagin or pharmaceutically acceptable derivatives thereof and (b) pharmaceutically acceptable excipients for the manufacture of a medicament for treating disorders or conditions in which it predominates, including acute and chronic (inflammatory, neuropathic and other).

According to another embodiment of the present invention there is provided a method for treating or preventing acute and chronic pain comprising administering to humans or animals an effective amount of detelokinobufagine or a pharmaceutically acceptable derivative thereof.

According to another embodiment of the present invention there is provided a method for treating acute screen pain which comprises administering to humans or animals a pharmaceutical composition comprising an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable excipients.

The present invention also discloses a pharmaceutical composition comprising telokinobufagine, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable excipient.

The pharmaceutical composition of the present invention may be administered by the following routes of administration: oral, sublingual, nasal, rectal, intragingival, intravenous, intramuscular, intraarticular, subcutaneous, inhalation, transdermal, topical and spinal (particularly subarachnoid and epidural). It is oral.

The term "animals" as used in the present invention refers to domestic animals, captive or non-wild animals and laboratory animals.

The term "effective amount" as used in the present invention refers to an amount of telokinobufagine that provides the desired analgesic activity when administered at the appropriate dose for each route of administration.

The term "pharmaceutically acceptable excipients" as used herein refers to ingredients that are compatible with other ingredients and are not harmful to humans or animals.

The term "pharmaceutically acceptable derivatives" as used herein refers to enantiomers, polymorphs, pseudopolymorphs (hydrates and solvates), prodrugs such as carbonates, carbamates, phosphates, phosphonates, glycosides, sulfates, sulfonates, ethers and esters. .

The term "treating" as used in the present invention refers to reversing, alleviating, inhibiting, preventing, or decreasing pain progression in humans or animals. The term "treatment" as used in the present invention refers to the treatable condition as defined above and prevention. The term "prevention" as used in the present invention refers to the preventative act which is related to the term "preemptive analgesia". This term refers to the preventive treatment of pain before it occurs. Such procedure has been used extensively after operations, where the attending physician knows that pain will occur after anesthesia has ceased. It is also used to treat conditions such as burns and chronic conditions such as cancer diseases, in which the doctor prescribes analgesic to be taken at fixed times and not just when the patient complains of pain. Preemptive analgesia aims to provide patient comfort by preventing the onset of pain and to prevent neuronal hyperexcitability and sensitization that occur in response to peripheral nociceptive stimuli. Central sensitization, when established, is difficult to suppress, and may be associated with the onset of neuropathic pain [Woof, C.J.1983. Evidence for a central component of post-injury painhypersensitivity. Nature, 686-688; Wall, P.D. 1988. Theprevention of postoperative pain. Pain].

The term "treating predominant disorders or conditions, including acute and chronic pain", as used in the present invention, refers to pain caused by selected conditions of the group below: congenital or genetic disorders; trauma, surgery or burns; infectious and / or parasitic disease; metabolic disease; inflammation autoimmune disease; cancer disease; poisoning, metabolic disorders and diseases; degenerative conditions (nervous system and other systems); dysfunctional symptoms, psychological changes.

Disorders or conditions in which pain predominates, including acute and chronic pain, "as used in the present invention, are selected from the non-limiting group consisting of tissue (soft and peripheral) injuries such as musculoskeletal and traumatic injuries, injuries of the nervous system of traumatic origin, including root or spinal cord injuries, and mechanical, radiation, surgical, thermal, chemical or electrical burn injuries, pain due to oncologic cause or not oncological evolution that presents acute or chronic evolution, due to somatic mechanism (for example nociceptive ) neuropathic or psychogenic; osteoarthritis, arthritis, osteoarthritis; musculoskeletal pain, particularly apostrum; orofacial pain, for example toothache; primary and secondary headache, including migraine; abdominal pain; pain due to cancer including benign and malignant neoplasias, for example cancer pain postoperative pain; pain due to neurodegenerative diseases, including multiple sclerosis and amyotrophic lateral sclerosis; pain resulting from neuropathies associated with degenerative diseases, including diabetes and also intervertebral dodial hernia; pain due to metabolic disorders, including diabetes mellitus, hypo or hyperthyroidism; pain due to repetitive strain injuries (RSI); pain from congenital or genetic disorders; pain from infectious or parasitic diseases, including leprosy, herpes zoster, acquired immunodeficiency syndrome, intoxication, including heavy metals; afferentation pain, central pain, phantom limb pain, causalgia, myelopathic pain, complex regional pain syndrome, amputation stump pain, reflex sympathetic dystrophy, postherpetic neuralgia, ischemic neuropathy, polyarthritis, post-radiotherapy pain, polyneuropathies , multiple mononeuritis, infectious and neurodegenerative myelopathy, toxic neuropathy, vasculitis and syringomyelia.

Description of the figures

The following is a description of the accompanying figures for a better understanding and illustration of the present invention.

Figure 1: Analgesic effects of orally administered telokinobufagin (curved / response) by abdominal writhing test in mice.

Figure 2: Comparison of the effects of telokinobufagin (10mg / kg, vo) with morphine sulfate "Morf" (10 mg / kg, ip), sodium diclofenac "DICL" (50 mg / kg, ip), sodium dipyrone "DIP" (50 mg / kg, ip) "COD" codeine phosphate (20 mg / kg, ip), by abdominal writhing test in mice.

Figure 3: Effects of naloxone (5 mg / kg, i.p.) on the analgesic action of telokinobufagin (10 mg / kg, v.o.), through abdominal writhing in mice.

Figure 4: Analgesic effects of orally administered telokinobufagin (Curvadose / response) in formalin-phase 1 mice.

Figure 5: Comparison of the effects of telokinobufagin (10mg / kg, vo) with morphine "Morf" sulfate (10 mg / kg, ip), "DICL" sodium diclofenac (50 mg / kg, ip), "DIP" sodium dipyrone (50 mg / kg, ip) and "COD" codeine phosphate (20 mg / kg, ip) in the formalin test phase 1 (4%) in mice.

Figure 6: Effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagin (5 mg / kg) in testeda ** formalin phase 1.

Figure 7: Analgesic effects of orally administered telokinobufagin (curved / response) in formalin-phase 2.

Figure 8: Comparison of the effects of telokinobufagin (5mg / kg, vo) with morphine "Morf" sulfate (10 mg / kg, ip), "DICL" sodium diclofenac (50 mg / kg, ip), "DIP" sodium dipyrone (50 mg / kg, ip) and codeine phosphate "COD" (20 mg / kg, ip), in Phase 2 of the formalin test.

Figure 9: Effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagin (5 mg / kg) on formalin testase phase 2.

Figure 10: Analgesic effects of orally administered telokinobufagin (lmg / kg, 2.5mg / kg and 5mg / kg) on the Hot Plate test.

Figure 11: Effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagin (5 mg / kg) on the Hot Plate test.

Figure 12: Effects of telokinobufagin (1mg / kg, 2.5mg / kg and 5mg / kg) on the tail flick test.

Figure 13: Effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagin (5 mg / kg) on the tail flick test.

For the figures below: PD = right paw before sciatic nerve ligation surgery; PDtest = right paw after sciatic nerve ligation surgery.

Figures 14 and 15: Effects of telokinobufagin (curved / response) on hyperalgesia via intraperitoneal (14) and oral (15) pathways, respectively.

Figure 16: Comparison of the analgesic effects of datelokinobufagine on ip (5mg / kg) and vo (20 mg / kg) hyperalgesia with the following analgesics: diclofenac sodium nDICL "(50 mg / kg, ip), nMorf demorphine sulfate "(10 mg / kg, ip)," COD "codeine phosphate (30 mg / kg, ip), nDIP sodium dipyrone" (300 mg / kg, ip), nAMT "amitriptyline hydrochloride" (30 mg / kg), carbamazepinanCBZ "(60 mg / kg) and nPRED prednisone" (10 mg / kg).

Figure 17: Effects of naloxone (5 mg / kg, i.p) on the analgesic action of telokinobufagin (5 mg / kg) on the hyperalgesia test.

Figures 18 and 19: Duration of analgesic effects of telokinobufagin on hyperalgesia, via intraperitoneal 5mg / kg (18) and oral, 20mg / kg (19), respectively.

Figures 20 and 21: Effects of telapinobufagin on allodynia (dose / response curve) via intraperitoneal (20) and oral routes (21), respectively.Figure 22: Comparison of the analgesic effects of datelokinobufazine on ip allodynia (5mg / kg ) ev.o (20 mg / kg) with the following analgesics: diclofenacosodic "DICL" (50 mg / kg, ip), morphine sulfate "Morf" (10mg / kg, ip), codeine phosphate "COD" ( 30 mg / kg, ip), DIP sodium dipyrone (300 mg / kg, ip), deamitriptyline hydrochloride "AMT" (30 mg / kg) and carbamazepine "CBZ" (60 mg / kg) and prednisone "PRED" (10 mg / kg).

Figure 23: Effects of naloxone (5 mg / kg, i.p) on the analgesic action of telokinobufagin (5 mg / kg, i.p.) on the dealodynia test.

Figures 24 and 25: Duration of the analgesic effects of telokinobufagin on allintraperitoneal allodynia, 5 mg / kg (24) and oral, 20 mg / kg (25), respectively.

Figure 26: Effects of ethanol (control), bupivacaine etelokinobufagine on the cardiac contraction force (inotropism) of rats at different concentrations (10 "7 to 10" 4M).

Figure 27: Polygraph plot representing the effect of bupivacaine in the left atrium of rats. The arrows indicate the moment of addition of bupivacaine (IO-4M).

Figure 28: Effects of telokinobufagin on cardiac contraction force (inotropism) during 12 minutes of observation using telokinobufagin at the concentration of 10 -3 M.

Figure 29: Effects of ethanol, bupivacaine and telokinobufagin on the frequency of spontaneous contraction of the right atrium in different concentrations (IO-7 to 10 "4M) .Figure 30: Polygraph plot showing the effect of bupivacaine in the right atrium of rats. arrows indicate the moment of addition of bupivacaine (10 4 M). BS-basal The features of the present invention will become apparent from the following detailed description.

Detailed Description of the Invention

The present invention is directed to the use of detelokinobufagin, or pharmaceutically acceptable derivatives thereof, in the manufacture of a medicament for treating or preventing pain. More specifically, the present invention describes the use of telokinobufagin for treating or preventing acute and chronic pain in humans or animals.

According to another embodiment of the present invention there is provided a method for treating or preventing acute and chronic pain comprising administering to humans or animals an effective amount of detelokinobufagin; or pharmaceutically acceptable derivatives thereof.

According to another embodiment of the present invention there is provided a method for treating acute screen pain, comprising administering to humans or animals a pharmaceutical composition comprising an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable excipients.

Telokinobufagin according to the following results has a higher potency than the damorphine shown when the comparison of results is performed considering the molar masses of the substances, without, however, presenting the known side effects of opioids. Additionally, the results of the present invention show that analgesic effects are independent of the opioid system.

The results obtained for analgesia in response to acute pain as well as chronic neuropathic pain were compared with several other conventional donor treatments using drugs such as morphine sulfate, decodein phosphate, sodium diclofenac, sodium dipyrone, prednisone, amitriptyline hydrochloride and carbamazepine. Telokinobufagin showed superior efficacy.

The results of the present invention show that TCB does not act with the same mechanism of action of opioids since naloxone, a specific morphine-derived antagonist, was unable to reverse its analgesic actions. Thus, treatment of acute and chronic pain with TCB is achieved without causing the side effects commonly seen with conventional opioid treatments, such as: constipation, physical addiction, respiratory depression, gastrointestinal injuries, nephrotoxicity, inhibition of platelet aggregation, incidence of related adverse effects. central nervous system (blurred vision, dizziness, headache, mental confusion, nausea, etc.).

It has also been found from the in vivo and in vitro test results that telokinobufin shows no signs of cardiac toxicity in isolated rat atrium preparations.

Another embodiment of the present invention relates to a pharmaceutical composition containing an effective amount of telokinobufagin, or pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable excipients. The pharmaceutical composition according to the present invention comprises from 0.1% to 99% w / w detelokinobufagine, or pharmaceutically acceptable derivatives thereof.

Telokinobufagin may be of natural or synthetic origin, and may be in its various possible pharmaceutically acceptable forms of enantiomers, polymorphs, pseudopolymorphs (hydrates and solvates), prodrugs such as carbonates, carbamates, phosphates, phosphonates, glycosides, sulfates, sulfonates, ethers and esters.

An example of derivatives suitable for the invention is an ester formed from the reaction of a datelokinobufagine hydroxyl group (examples: C3-0H, C5-0H or C14-0H) with a carboxylic acid, for example alkyl Cn-CO2H and HO2C- (CH2) n-CO2H (where η is 1-10), and phenyl-CH2-CO2H. An example for ether formation is the reaction of a hydroxyl group (examples: C3-OH, C5-0H or C14-0H) with alkyl halides (Cn-X, where η is 1-10 and X is Cl, Br, I F) and / or alkyl or arylsulfonates.

The skilled artisan will recognize that atelokinobufagin exhibits chirality, and therefore contain atoms which may be in a particular geometrical and stereochemical configuration, giving rise to conformational isomers and stereoisomers. All isomers and mixtures thereof are included in this invention.

According to the present invention, the administration of detelokinobufagin or the same-containing pharmaceutical compositions may be by the oral, sublingual, nasal, rectal, intragival, intravenous, intramuscular, intraarticular, subcutaneous, dermal (eg, patch or transdermal patch) administration routes. ), inhalation, transdermal, topical and spinal (subarachnoid and epidural), not limited to these, and the route of administration is preferred over the oral route.

Pharmaceutically acceptable excipients are selected according to the final presentation of the composition of the present invention which may be in the form of capsules or tablets for oral administration, nasal administration solutions, injectable solution for intramuscular, intravenous, cutaneous or subcutaneous administration, lotion, cream, gel or ointment for topical administration. Methods for preparing various pharmaceutical compositions are well known, or will be apparent in light of the present invention, to the skilled artisan. For example, see Remingtons Pharmaceutical Sciences, 18th ed. (nineteen ninety).

The examples cited below are merely illustrative and should be used solely for a better understanding of developments in the present invention, but should not be used to limit the objects described.

In vivo tests for acute pain:

The in vivo testing methods for acute pain used included the writhing test, formalin test, hot plate test, and tail pull test.

The different assays were performed at various TCB doses per v., Using at least 6 cadadose animals. Statistical analysis of the results was performed using ANOVA, followed by the TUKEY test (p <0.05).

Route of administration: Telokinobufagin was orally administered and dissolved in 40% dimethyl sulfoxide (DMSO) in water. The animals were fasted for 12 hours before the start of the experiments. Other drugs, morphine sulfate, codeine phosphate, diclofenac sodium, dipyrone sodium, prednisone, amitriptyline hydrochloride and carbamazepine were administered intraperitoneally (i.p.).

Animals: About 500 Swiss (Mus musculus) mice were used.

Example 1 - Writhing Test

Mouse Writhing Test: discrimination between central and peripheral analgesic activities. This test is based on the intraperitoneal administration of serous membrane-irritating substances, for example acetic acid, causing stereotyped behavior in the mouse, which is mainly characterized by abdominal contractions, accompanied by stretching of the hind paws. This behavior is considered to be of reflective origin and evidence of Visceral Pain, similar to that resulting from a peritonitis picture. Although this test is not very specific, it is extremely sensitive, being one of the most used tests for screening of new analgesics. Since the writhing test allows to evaluate the antinociceptive activity of both central and peripheral acting substances, when combined with a specific central action test (hot plate), it can be demonstrated whether the level of action of a given drug is central and peripheral or exclusively peripheral. , or exclusively central. Control groups received the same volume of vehicle (10 ml / kg) used to dilute the test compounds orally 1 hour before the test, while the treated groups received increasing doses of the tested substances under these same conditions. drug administration by you and then, five minutes after acetic acid injection (0.6%, via i.p.), the animals were observed, cumulatively quantifying the number of abdominal contortions over a period of 20 minutes. Anticociceptive activity was determined on the basis of the inhibition of the number of writhes of treated animals relative to the number of writhes of the control animals. Results are shown in Figures 1-3.

Figure 1 shows the analgesic effects of datelokinobufagine (dose / response curve) administered via the oral writhing test in mice. This figure demonstrates that telokinobufagin had significant antinociceptive effects in this test, even blocking, at the dose of 10 mg / kg, about 90% of the writhes compared to the control group (p <0.001).

Figure 2 shows the comparison of the effects of datelokinobufagin (10 mg / kg, vo) with morphine sulfur (10 mg / kg, ip), sodium diclofenac "DICL" (50 mg / kg, ip), sodium dipyrone "DIP" ( 50 mg / kg, ip) "COD" codeine phosphate (20 mg / kg, ip) by mouse writhing test shows that telokinobufagin had an antinociceptive effect, mol to mol, about 40%. greater than that damorphine intraperitoneally and much higher than the other analgesics used in the test that were also administered intraperitoneally (p <0.05).

Figure 3 shows the effects of naloxone (5 mg / kg) on the analgesic action of telokinobufin (10 mg / kg) through the mouse writhing test. This figure shows that naloxone at a dose of 5 mg / kg , ip, blocked the effects of morphine (p <0.05) administered at a dose of 10 mg / kg, ip, but did not block the analgesic effects of telokinobufin, eg. at a dose of 10mg / kg.

Example 2- Formalin Test

Assessment of Neurogenic and Inflammatory Acute Pain. Intraplantar formalin injection test (4%) induces noanimal licking or twitching reactions. This test allows us to evaluate two types of pain: one of neurogenic origin, as a result of the direct stimulation of nociceptive neurons observed in the first 5 minutes after formalin administration, and another of inflammatory origin, characterized by the release of inflammatory mediators, observed between 15 and 30 minutes after injection. formalin and representing the aching tonic response. The test was performed at room temperature of 24-25 ° C in the absence of experimental factors that could affect peripheral blood flow, due to the high response sensitivity in the second (late) phase. The control groups received the same vehicle volume (10 mL / kg) used to dilute the test compounds orally 1 hour before the test, while the treated groups received, under the same conditions, increasing doses of the substances to be tested. The antinociceptive activity was then determined on the basis of decreased licking time or paw contraction of animaistrates in relation to the same responses as group control animals.

The results of the formalin test are shown in Figures 4-9.

Figure 4 shows the analgesic effects of oral datelokinobufagin (dose / response curve) administered in phase 1 of the formalin test in mice. This figure shows that telokinobufagin had significant antinociceptive effects in this test, even blocking at 10mg dose. / kg, about 80% of the lipids in relation to the control group (p <0.005).

Figure 5 shows the comparison of the effects of datelokinobufagine (10 mg / kg, vo) with morphine sulfate "Morf" (10 mg / kg, ip), diclofenac sodium "DICL" (50mg / kg, ip), sodium dipyrone wDIP "(50 mg / kg, ip)" COD "codeine ephosphate (20 mg / kg, ip), in phase 1 with formalin test (4%) in mice. This figure shows that telokinobufagin by itself had an antifungal effect. mol to mol, about 40% greater than that of intraperitoneally morphine and far superior to those of the other analgesics used in this test (p <0.05). Figure 6 shows the effects of naloxone (5 mg / kg) on analgesic action of telokinobufagin (5 mg / kg) in formalin test phase 1. This figure shows that analoxone at a dose of 5 mg / kg ip blocked the effects of 10 mg / kg (p <0) , 05), but did not block the analgesic effects of telocibufagine, for a 5 mg / kg dose.

Figure 7 shows the analgesic effects of datelokinobufagine (dose / response curve) on phase 2 of orally administered testeda formalin. This figure shows that telokinobufagin had significant antinociceptive effects in this test, even blocking, at 10 mg / kg, about 100% of the licks in relation to the control group (p <0.0001).

Figure 8 shows the comparison of the effects of datelokinobufagine (5 mg / kg, vo) with morphine sulfate "Morf" (10 mg / kg, ip), sodium diclofenac "DICL" (50mg / kg, ip), sodium dipyrone "DIP "(50 mg / kg, ip) Codeine Ephosphate" COD "(20 mg / kg, ip) in Phase 2 with formalin. This figure shows that atelokinobufagin, for you. showed an antinociceptive effect, mol to mol, about 58% greater than that of morphine intraperitoneally and much higher than that of the other analgesics used in this test (p <0.05).

Figure 9 shows the effects of naloxone (5 mg / kg) on the analgesic action of telokinobufin (5 mg / kg) in formalin test phase 2. This figure shows that analoxone at a dose of 5 mg / kg, i.p., blocked the effects of amorphine (p <0.05) administered at the dose of 10 mg / kg, but did not block the analgesic effects of telokinobufagin, perhaps. at a dose of 5mg / kg. Example 3- Hot Plate Test

Determination of Central Analgesic Activity. This test consists of placing the animals on a hot plate (52 +0.5 ° C) and observing how long it takes to manifest a response, particularly licking or jumping. Both reactions are considered integrated superspinous (brain) responses. Control groups received the same vehicle volume (10 mL / kg) used to dilute the test compounds orally 1 hour before the test, while the treated groups received, under these same conditions, increasing doses of the substances to be tested. The antinociceptive activity was determined based on the increase of the latency time to paw the feet or jump of the treated animals in relation to the same responses of the control animals. The results are shown in Figures 10 and 11.

Figure 10 shows a dose / response curve of the analgesic effects of orally administered telokinobufagin (1 mg / kg, 2.5 mg / kg and 5 mg / kg) in the Hot Plate test. This figure demonstrates that telokinobufagin had significant antinociceptive effects in this test, increasing response by about 450% at the dose of 5 mg / kg, v.o., relative to the control group (p <0.001).

Figure 11 shows the effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagin (5 mg / kg) on the hot plate. This figure demonstrates that naloxone at a dose of 5 mg / kg, ip, blocked the effects of amorphine (p <0.05) administered at the dose of 10 mg / kg, but did not block the analgesic effects of telokinobufagin, for each dose of 5mg / KgExample 4 - Tail Withdrawal Test

Determination of Medullary Analgesic Activity.

We opted for the test variant which consists of applying thermal radiation through a filament that is heated for a few seconds to 70 ° C in contact with a small surface of the animal's tail. This thermal stimulus elicits a tail-tailed reaction of the control animals through a rapid, spinal reflex reflex movement after a time ranging from 2 to 10 seconds. Increased tail removal time is interpreted as an analgesic action. Control groups received the same vehicle volume (10 mL / kg) used to dilute the test compounds orally 1 hour before the test, while the treated groups received, under these same conditions, increasing doses of the substances to be tested. The antinociceptive activity was determined based on the increase in the tail-off latency time of the treated animals in relation to the same responses of the control group animals. The results of this test are shown in Figures 12 and 13.

Figure 12 shows the effects of telokinobufagin (1mg / kg, 2.5mg / kg, and 5mg / kg) on the Tail Flick test. This figure shows that telokinobufagin had, at doses of 2.5 and 5 mg / kg, significant antinociceptive effects in said test (p <0.05).

Figure 13 shows the effects of naloxone (5 mg / kg) on the analgesic action of telokinobufagine (5 mg / kg) on the tail flick. This shows that naloxone at a dose of 5 mg / kg ip blocked the effects of morphine (p <0.05) administered at a dose of 10 mg / kg but did not block the analgesic effects of datelokinobufagin at 5 mg dose. / KgIn vivo tests for chronic pain:

Example 5 - Chronic Constructive Injury Model (ChronicCOZistrictive injury, CCI)

Recently, several animal models of neuropathic pain have been developed in rats. According to the method described by Bennett [Bennett. G. J; Xie, Y.K. 1998. A peripheralmononeuropathy in rat that produces disorders of pain sensations. Pain, 33: 87-107], under anesthesia, the rat is positioned prone and an incision is made in the skin along the thigh. The fascia between the buttocks and the femoral biceps muscle is dissected and the right common nerve cell is exposed at the median level of the thigh. Near this trifurcation, the nerve is carefully dissected from the surrounding tissue for a distance of about 8mm, at which it is performed. test.

In this experimental model of neuropathic pain were measured: a) Allodynia: pain in response to non-injurious stimuli; b) Hyperalgesia: exaggerated pain in response to injurious stimuli. Peripheral neuropathy was produced by loose sciatic nerve ligation (Chronic constrictiveinjury, ICC) according to the method described by Bennett andXie (1988). Adult rats were anesthetized with sodium compentobarbital (40 mg / kg, i.p.) and their sciatic nerves exposed through dissection of the bicepsfemural muscle. On one side, four loose constrictive ligatures with chromium (4-0) suture were performed around the nerve, spaced approximately 1 mm apart. On the contralateral side, all steps of the surgical process were performed, such as exposure and handling of the nerve, except the ligatures. To minimize technical variations, all of these procedures were performed by one person. At the end, a single dose of antibiotic was administered (Ampicillin 8000 u / rat, Sigma, St Louis, MO). Before the tests were performed, a time of about 5 days was used for the recovery of animals. Allodynia Determination. Using the von Frey equipment, the Chaplan method [Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L. 1994.

Quantitative assessment of tactile allodynia in the rat pa J.J Neurosci Methods. 53: 55-63]. The response time to the tactile stimulus applied to the posterior paw dorato was measured.

Determination of hyperalgesia. The rat paw depression test method was used by the Randall & Sellito method [Randall, L.O., Selittof J.J. 1957. A method for measurementof analgesic activity on inflamed tissue. Arch IntPharmacodyn Ther 111: 409-419]: The force was continuously applied to the back of the paw (16 g / s) and interrupted when withdrawal occurs. The results of the tests performed through the chronic constrictive injury model are shown in Figures 14-25.

Figures 14 and 15 show the effects of datelokinobufagine (dose / response curve) on hyperalgesia via the intraperitoneal and oral routes, respectively. These figures show that telokinobufagin had significant antinociceptive effects in these tests, increasing responses approximately 9 and 3.9 times in relation to the control group (PDtest) by ip (5 mg / kg) (p <0.001) and ν.o . (20 mg / kg) (p <0.05), respectively.

Figure 16 shows the comparison of analgesic effects of telokinobufugin, ip (5 mg / kg) and ev.o (20 mg / kg), with the following analgesics on hyperalgesia: sodium diclofenac "DICL" (50 mg / kg, ip) , morphine sulfate "Morf" (10 mg / kg, ip), decodein phosphate uCOD "(30 mg / kg, ip), sodium dipyrone" DIP "(300mg / kg, ip), amitriptyline hydrochloride" AMT "(30 carbamazepine "CBZ" (60 mg / kg) and prednisone "PRED" (10mg / kg) This figure shows that telokinobufagin via ν.o and ip had antinociceptive effects, molamol, about 900%. greater than that of ip morphine, and also significantly higher than that of other analgesics used in the same test (p <0.05), however, the effect of TCB when compared mol to mol, as with that of intraperitoneal morphine Figure 17 shows the effects of naloxone (5 mg / kg, ip) on the analgesic action of telokinobufagin (5 mg / kg) on the hyperalgesia test. shows that naloxonan dose of 5 mg / kg ip blocked the effects of morphine (p <0.05) administered at the dose of 10 mg / kg but did not significantly block the analgesic effects datelokinobufagin ip at 5 mg / kg.

Figures 18 and 19 show the duration of analgesic effects of telokinobufagine on hyperalgesia, i.p and oral, respectively. These figures show the analgesic effects of telokinobufagines on i.p (5mg / kg) and oral (20mg / kg) hyperalgesia lasted about 6 hours, with an estimated half-life of 3 hours (p <0.05).

Figures 20 and 21 show the effects of datelokinobufagin on allodynia by oral intraperitoneal routes, respectively. These figures show that atelokinobufagin had significant antinociceptive effects in this test, reaching allodynia blockade at doses of 5 mg / kg i.p and 20 mg / kg per v.o (p <0.05).

Figure 22 shows the comparison of analgesic effects of telokinobufagin, ip (5mg / kg) and vo (20 mg / kg), with the following analgesics on allodynia: diclofenac sodium "DICL" (50 mg / kg, ip), morphine sulfate "Morf" (10 mg / kg, ip), codeine phosphate "COD" (30 mg / kg, ip), sodium dipyrone "DIP" (300 mg / kg, ip), deamitriptyline hydrochloride "AMT" ( 30 mg / kg), carbamazepine "CBZ" (60 mg / kg) and prednisone "PRED" (10 mg / kg). This figure shows that telokinobufin via the vo and ip routes completely block allodynia at doses of 5 mg / kg ip and 20 mg / kg per vo (p <0.05) at lower doses than amitriptyline (30 mg / Kg) and carbamazepine (60 mg / kg) administered via p (p <0.05). The results showed that TCB, by both i.p. and v.o., completely reversed the dealodynia picture (in relation to PD), while i.p. morphine reversed only about 50% of allodynia (in relation to PD).

Figure 23 shows the effects of naloxone (5 mg / kg, i.p) on the analgesic action of telokinobufagin (5 mg / kg, i.p) on the allodynia test. This figure demonstrates that analoxone at a dose of 5 mg / kg ip blocked the effects of damorphine (p <0.05) administered at the dose of 10 mg / kg but did not significantly block the analgesic effects of datelokinobufagine ip at the dose. of 5 mg / kg.

Figures 24 and 25 show the duration of the analgesic effects of telokinobufagin on oral i.p and oral allodynia, respectively. These figures show that the analgesic effects of telokinobufagin on i.p (5mg / kg) and orally (20 mg / kg) allodynia lasted about 6 hours, with an estimated half-life of 3 hours (p <0.05).

Live xn assays to assess cardiotoxicity:

Example 6 - Effect of telokinobufagin on isolated atrial rat inotropism.

In order to study the possible deleterious effects of telokinobufagin noxiousness, the isolated rat atrium model was initially used to assess the cardiac contraction strength (inotropism). The left atrium was used to study inotropic activity. Data were expressed as contraction force (g tension) and compared with the results obtained with bupivacaine.

The application of square pulses (5 V, 5 ms, 2 Hz) in the left atrium with a frequency of 120 bpm was sufficient to produce a cardiac contraction force of 0.63 ± 0.09g of tension. The response in contractility does not change significantly when the conditions to which the left atrium was subjected to superinfusion were maintained. The results of these tests are shown in Figures 26-28.

Figure 26 shows that in the presence of telokinobufagine, no significant change in cardiac contraction strength compared to control at concentrations of 10 "7 to 10" 4 M was observed. However, bupivacaine at a concentration of 3 χ 10 "5 M decreased significantly. cardiac decontraction force to 0.13 ± 0.06 g of tension, and IO "4 M concentration totally blocked cardiac decontraction force to zero (p <0.05, Dunnett's test). Data are expressed as mean ± SEM (n = 6). Anova eDunnett as post hoc test, p <0.005. a vs control, b vsbupivacaine.

In Figure 27 is shown an empirically recorded plot representing the effect of bupivacaine on the left atrium of rats. The arrows indicate the moment of addition of bupivacaine (10 "4 M).

In order to find a toxic dose of datelokinobufagin, a concentration of 10 "3 M for 12 min instead of 5 min was used (Figure 28). However, even at this concentration, telokinobufagin also did not produce a significant change in the contraction strength ( p> 0.05, Dunnett's test).

Example 7 - Effect of telokinobufagin on isolated rat atrial chronotropism.

In order to study the possible deleterious effects of nocturnal effects, the rat atrial isolate model was subsequently used to assess the frequency of cardiac contraction (chronotropism). The rat right atrium was used to study chronotropic activity. The values of spontaneous mechanical activity of the right atrium were expressed in frequency of contractions or beats per minute (bpm). Heart rate was 298 ± 50contractions per minute in the right atrium of the rat. The frequency of spontaneous cardiac activity does not change significantly when the conditions to which the right atrium was subjected to overfusion were maintained.

Figure 2 9 shows that in the presence of telokinobufagin no significant change was observed in spontaneous cardiac activity compared to control at concentrations of 10-7M to 10 "4 M (n = 6; ρ> 0.05, Dunnett test). However, bupivacaine at a concentration of 3 χ10 "5 M significantly decreased the frequency of cardiac spontaneous activity to 9.6 ± 2.4 bpm, and at a concentration of 10" 4 M totally blocked the heart rate to zero (p <0.05; Figure 29 shows the effect of dabupicavain and telokinobufagin on the frequency of spontaneous contractions of the right atrium of rats at different concentrations (10 "7M to 10" 4M). Data are expressed as mean ± SEM (n = 6). Anova and Dunnett as post hoc test, p <0.005 a vs control, b vs bupivacaine.

In Figure 30 is shown an empirically recorded plot depicting the effect of bupivacaine on the right atrium of rats. Arrows indicate the time of the addition of bupivacaine (IO "4 M). BS-basalIn vitro tests to assess cardiotoxicity:

Example 8 - Patch Clairtp Assay

The in vitro effects of telokinobufagin on ionic channels were evaluated at room temperature using PatchXpress® (Model 7000A, Molecular Devices, Union CityCA) - automatic parallel patch clamp system. The studies were conducted according to conventional procedures.

To perform the tests, a telocinobufin 30 µ 0 stock solution in 0.3% DMSO in buffer solution (HBPS) was prepared. The test was evaluated using telokinobufagin15 μΜ in 2 cells (n> 2). The exposure duration of each channel was 5 minutes. Telokinobufagin (> 95% purity) is water-insoluble, soluble in 50% (v / v water) DMSO, 100% ethanol and pure chloroform. Each recording hKvLQTl / hminK and hCavl.2 (h = human) ended with a maximum dose of the positive control (chromanol 300 μΜ enifedipine 10 μΜ, respectively). Any left currents following exposure to this dose have been digitally subtracted to eliminate the contribution of any endogenous current. For this reason, some negative results are obtained for some measures.

Results for telokinobufagin are shown in Table 2. The positive control shown in Table 3 confirms the sensitivity of the test system for ion channel inhibition. Significance criteria: ^ 20% inhibition.

Table 2 - Result of inhibition of pelatelokinobufagin ion channels.

<table> table see original document page 36 </column> </row> <table> <table> table see original document page 37 </column> </row> <table>

Table 3 - Positive control for ionic decay inhibition tests.

<table> table see original document page 37 </column> </row> <table>

Results of the mean inhibition percentage described in Table 2 show that the various evaluated ion channels were not inhibited by telokinobufagin. These results corroborate the results of the in vivo tests performed to evaluate the effect of telokinobufagin on isolated atrial rat atrial chronotropism and chronotropism, thus, evidencing the absence of pelatokinobufagin cardiotoxicity.

Example 9 - Radioligand Binding Assay (ra.dioliga.ndbinding-assays)

The methods employed were adapted from scientific literature to maximize their reliability and reproducibility. Reference standards were used as an integral part of each assay to ensure the validity of the results obtained.

This study aimed to evaluate the activity of datelokinobufagin [Purity ^ 95%] on opioid receptors. Currently these receptors are classified into three groups, μ, δ, k. The results of these tests are shown in Table 4 and the methods used are described below:

Opioid Receptor δ (OP1, DOP)

Source: Recombinant Human CHO Cells

Binder: [3H] Naltrindola 0.9 nM

Vehicle: DMSO 1%

Incubation time / temp: 2 hours at 25 ° C.

Incubation Buffer: 50mM Tris-HCl, pH 7.4, 5mM MgCl2

Non-specific binder: Naloxone 10μΜ

KD: 0.49nM *

Bmax: 8.6 pmole / mg Protein

Specific binding: 80%

Quantitative method: Radioligant binding (RadioligantBinging)

K opioid receptor (0P2, KOP)

Source: Recombinant Human HEK-293 Cells

Binder: [3H] Diprenorphine 0.6 nM

Vehicle: DMSO 1%

Incubation time / temp: 60 minutes at 25 ° C.

Incubation Buffer: 50 mM Tris-HCl, pH 7.4

Non-specific binder: Naloxone 10μΜ

K0: 0.4 nM

Braax: l.lpmole / mg Protein

Specific binding: 90%

Quantitative method: Radioligant binding (RadioligantBinging)

Μ opioid receptor (OP3, M0P)

Source: Recombinant Human CH0-K1 Cells

Binder: [3H] Diprenorphine 0.6 nM Vehicle: 1% DMSO

Incubation time / temp: 60 minutes at 25 ° C.

Incubation Buffer: 50 mM Tris-HCl, pH 7.4

Non-specific binder: Naloxone ΙΟμΜ

K0: 0.41 nM

Bmax: 3.8 pmole / mg Protein

Specific binding: 90%

Quantitative method: Radioligant binding (RadioligantBinging)

Table 4 - Opioid receptor TCB activity test

<table> table see original document page 39 </column> </row> <table>

hum = human

* Significance criterion or significant response ^ 50% maximal stimulation or inhibition.

the opioid receptor selective N-methyl-N- (7- (1-pyrrolidinyl) -1-oxaspiro (4,5) dec-8-yl) benzeneacetamidaligante k.

b [D-Ala2fNMe-Phe4, Gly-ol5] -enkephalin - highly selective agonist peptide]

The percent inhibition results described in Table 4 show that the μ, δ, k opioid receptors were not inhibited or stimulated by telokinobufagin. These results corroborate with the in vivo assays which showed that the effects of telokinobufagin are not reversed by naloxone. It is known then that its mechanism of action is not opioid and therefore it is expected that this compound will be devoid of the adverse side effects of opioids.

Claims (34)

1. Use of telokinobufagin, or pharmaceutically acceptable derivatives thereof, characterized in that it is in the manufacture of a medicament for treating or preventing acute or chronic odors in humans or animals. Use according to Claim 1, characterized in that it is useful for preemptive analgesia. Use according to claim 1, characterized in that the acute pain phage is of the neurogenic or inflammatory type. Use according to claim 1, characterized in that the pain phage is of oncological or non-oncological origin and is derived from a somatic, neuropathic or psychogenic mechanism. Use according to claim 1, characterized in that the chronic pain phage is neuropathic of the type hyperalgesia or allodynia. Use according to claims 1 to 5, characterized in that the pain is caused by conditions or diseases selected from the group consisting of congenital or genetic disorders; trauma, surgery or burns; infectious and / or parasitic disease; metabolic disease; inflammation; autoimmune disease; poisoning; metabolic disorders and diseases, neurodegenerative and degenerative conditions; dysfunctional pictures; psychological changes and benign and malignant neoplasms. Use according to claims 1 to 6, characterized in that acute or chronic pain is selected from one or more conditions or diseases of the group consisting of musculoskeletal, muscular, radicular, spinal cord injuries, mechanical injuries by radiation, surgery, burns. thermal, chemical or electrical; osteoarthritis, arthritis rheumatoid; musculoskeletal pain, particularly after trauma; toothache; headache, migraine; abdominal pain; cancer pain; postoperative pain; multiple sclerosis, amyotrophic lateral sclerosis; intervertebral dodial hernia; diabetes mellitus, hypo or hyperthyroidism; pain due to repetitive strain injuries (RSI); pain due to congenital or genetic disorders; pain due to leprosy, herpes zoster, AIDS acquired immunodeficiency syndrome, pain due to heavy metal poisoning; afferentation pain, central pain, phantom limb pain, causalgia, myelopathic pain, complex regional pain syndrome, myofascial pain syndrome, fibromyalgia, amputation stump pain, reflex dystrophias, postherpetic neuralgia, diabetic mononeuropathy, ischemic neuropathy, nodular polyaritis, post-radiotherapy pain, polyneuropathies, multiple mononeuritis, infectious and neurodegenerative myelopathies, toxic neuropathies, vasculitis and syringomyelia. Use according to claim 1, characterized in that the pharmaceutically acceptable derivatives of datelokinobufagin are enantiomers, polymorphs, pseudopolymorphs or prodrugs. Use according to claim 8, characterized in that the pellet of the pseudopolymorphs is hydrates or solvates. Use according to claim 8, characterized in that the prodrugs are carbonates, carbamates, phosphates, phosphonates, glycosides, sulphates, sulphonates, ethers or esters. Analgesic pharmaceutical composition characterized in that it contains an effective amount of telokinobufagine, or a pharmaceutically acceptable derivative thereof, and pharmaceutically acceptable excipients. Pharmaceutical composition according to Claim 11, characterized in that it comprises from 0.1% to 99% w / w of telokinobufagin or pharmaceutically acceptable derivatives thereof. Composition according to Claim 11, characterized in that the pharmaceutically acceptable telocinobufagin derivatives are enantiomers, polymorphs, pseudopolymorphs and prodrugs. Composition according to Claim 13, characterized in that the pseudopolymorphs are hydrates or solvates. Composition according to Claim 13, characterized in that the prodrugs are carbonates, carbamates, phosphates, phosphonates, glycosides, sulphates, sulphonates, ethers and esters. 16. Composition according to Claim 11, characterized in that it is administered to humans or animals by oral, sublingual, nasal, rectal, intragingival, intravenous, intramuscular, intraarticular, subcutaneous, inhalation, transdermal, topical, spinal routes. subarachnoid or epidural spinal cord. Composition according to Claim 16, characterized in that it is preferably administered orally. Use of the composition according to claim 11, characterized in that it is in the manufacture of a medicament for treating or preventing acute sonic pain in humans or animals. Use according to claim 18 characterized in that it is useful in preemptive analgesia. Use according to claim 18, characterized in that acute pain is neurogenic or inflammatory. Use according to claim 18, characterized in that chronic pain is neuropathic pain of the type hyperalgesia or allodynia. Use according to claims 18 to 21, characterized in that the pain is caused by conditions or diseases selected from the group consisting of congenital or genetic disorders; trauma, surgery or burns; infectious and / or parasitic disease; metabolic disease; inflammation; autoimmune disease; poisoning; metabolic disorders and diseases, neurodegenerative and degenerative conditions; dysfunctional pictures; psychological changes and benign and malignant neoplasms. Use according to claims 18 to 22, characterized in that acute or chronic pain is selected from one or more conditions or diseases of the group consisting of osteoarticular, muscular, radicular, medullary, mechanical, radiation, surgical, thermal, chemical or electrical burns; osteoarthritis, arthritis rheumatoid; musculoskeletal pain, particularly after trauma; toothache; headache, migraine; abdominal pain; cancer pain; postoperative pain; multiple sclerosis, amyotrophic lateral sclerosis; intervertebral dodial hernia; diabetes mellitus, hypo or hyperthyroidism; pain due to repetitive strain injuries (RSI); pain due to congenital or genetic disorders; leprosy pain, herpes zoster, AIDS-acquired immunodeficiency syndrome, pain due to heavy metal poisoning; afferentation pain, central pain, phantom limb pain, causalgia, myelopathic pain, complex regional pain syndrome, myofascial pain syndrome, fibromyalgia, amputation stump pain, reflex dystrophias, postherpetic neuralgia, diabetic mononeuropathy, ischemic neuropathy, nodular polyaritis, post-radiotherapy pain, polyneuropathies, multiple rhinoneuritis, infectious and neurodegenerative myelopathies, toxic neuropathies, vasculitis and syringomyelia. Use of the composition according to claim 18, characterized in that it is useful in veterinary medicine and namedicin. A method for treating or preventing acute or chronic pain, comprising the step of administering to humans or animals in need of such treatment an effective amount of detoxokinobufagin, or a pharmaceutically acceptable derivative thereof. 26. A method for treating or preventing acute screen pain, comprising the step of administering to humans or animals in need of such treatment, a pharmaceutical composition containing an effective amount of telocinobufagine, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable excipient. A method according to claim 25 or 26, which is useful for preemptive analgesia. A method according to claim 25 or 26, characterized in that the acute pain is neurogenic or inflammatory. A method according to claim 25 or 26, characterized in that the pain is of oncologic or non-oncologic origin and is derived from a somatic, neuropathic or psychogenic mechanism. A method according to claim 25 or 26, wherein the chronic pain is neuropathic hyperalgesia or allodynia type. A method according to claim 25 or 26, wherein the pain is caused by conditions or diseases selected from the group consisting of congenital or genetic disorders; trauma, surgery or burns; infectious and / or parasitic disease; metabolic disease; inflammation; autoimmune disease; poisoning; metabolic disorders and diseases, neurodegenerative and degenerative conditions; dysfunctional pictures; psychological changes and benign and malignant neoplasms. 32. A method according to claim 25 or 26, characterized in that pain is selected from one or more conditions or diseases of the group consisting of osteoarticular, muscular, radicular, spinal cord injuries, mechanical radiation injuries, surgical burns, chemical or electrical; osteoarthritis, rheumatoid arthritis; skeletal sleeplessness, particularly after trauma; headache, migraine; abdominal pain; cancer pain; postoperative pain; multiple sclerosis, amyotrophic lateral sclerosis; herniated disc intervertebral; diabetes mellitus, hypo or hyperthyroidism; pain due to repetitive strain injuries (RSI); pain due to congenital or genetic disorders; leprosy pain, herpes zoster, AIDS-acquired immunodeficiency syndrome, pain due to heavy metal poisoning; afferentation pain, central pain, phantom limb pain, causalgia, myelopathic pain, complex regional pain syndrome, myofascial pain syndrome, fibromyalgia, amputation stump pain, reflex dystrophias, postherpetic neuralgia, diabetic mononeuropathy, ischemic neuropathy, nodular polyaritis, post-radiotherapy pain, polyneuropathies, multiple mononeuritis, infectious and neurodegenerative myelopathies, toxic neuropathies, vasculitis and syringomyelia. Method according to claim 25 or 26, characterized in that the administration to human beings and animals is by oral, sublingual, nasal, rectal, intragival, intravenous, intramuscular, intraarticular, subcutaneous, inhalation, transdermal, topical administration. and subarachnoid and epidural spinal. The method according to claim 33, characterized in that the administration is preferably by oral route.